LL-37 is the 37-residue C-terminal fragment of human cathelicidin (hCAP18), the only cathelicidin-family peptide encoded in the human genome. It is a cationic, amphipathic alpha-helical peptide used in antimicrobial susceptibility testing, membrane biophysics and formyl peptide receptor 2 (FPR2) signalling assays. NuVion supplies LL-37 as a laboratory chemical for in vitro research use only.
Key facts
| Type/class | Synthetic peptide, human cathelicidin fragment, cationic antimicrobial peptide |
| Amino acid count | 37 |
| Sequence | LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES |
| Molecular formula | C205H340N60O53 |
| Molecular weight | 4493.3 g/mol |
| CAS number | 154947-66-7 |
| Synonyms | Cathelicidin LL-37, hCAP18 C-terminal peptide, CAMP antimicrobial peptide |
| Supplied form | Lyophilised powder in a sealed vial |
Structure and chemistry
The CAMP gene encodes an 18 kDa precursor, hCAP18, made up of a signal peptide, a conserved cathelin-like domain and a variable C-terminal antimicrobial domain. In neutrophils the precursor is stored in specific granules and the C-terminal domain is released by proteinase 3 after degranulation. In keratinocytes the same fragment is generated by kallikreins 5 and 7. The released 37-residue peptide starts with two leucines, which is where the name comes from. Transcription of CAMP is driven by a vitamin D response element in its promoter, so 1,25-dihydroxyvitamin D3 raises LL-37 expression in cultured keratinocytes and monocytes.
LL-37 carries five arginines and six lysines against two aspartates and three glutamates, giving a net charge of about +6 at neutral pH once the termini are counted. The sequence contains no cysteine, methionine or tryptophan, so there is no disulfide chemistry and the two main oxidation-prone residue types are absent. In water at low ionic strength the peptide is largely disordered. As salt concentration rises, or when it meets anionic lipid, detergent micelles or trifluoroethanol, it folds into an alpha-helix that runs from roughly residue 2 to residue 31, with the C-terminal tail (32 to 37) remaining flexible. The helix is amphipathic: hydrophobic residues (Phe, Ile, Leu, Val) line one face and the basic residues line the other. In aqueous buffer at physiological salt the hydrophobic faces pack together and LL-37 self-associates into oligomers, which is one reason its apparent activity depends on the medium used.
Shorter fragments are often run beside the parent peptide. KR-12 (residues 18 to 29) is the smallest fragment that keeps measurable antibacterial activity, and the all-D enantiomer D-LL-37 is used to separate chiral, receptor-dependent responses from achiral membrane effects.
Mechanism of action
Membrane interaction
The primary target of LL-37 is the microbial membrane. Electrostatic attraction to anionic headgroups (phosphatidylglycerol, cardiolipin, lipid A of lipopolysaccharide, lipoteichoic acid) concentrates the peptide at the bilayer surface, where it folds into the amphipathic helix and inserts its hydrophobic face into the acyl chain region. Above a threshold surface density the bilayer loses its barrier function through a toroidal pore or carpet-type mechanism, ions and small solutes leak, and the transmembrane potential collapses. Mammalian membranes, which present zwitterionic phosphatidylcholine and cholesterol on the outer leaflet, bind the peptide far more weakly, and this difference in headgroup charge is the basis of its selectivity in vitro. Activity against Gram-negative organisms is preceded by binding to lipopolysaccharide in the outer membrane, and the same lipid A binding blocks lipopolysaccharide from engaging LPS-binding protein, CD14 and TLR4 on monocytes in culture.
Receptor-mediated signalling
On mammalian cells LL-37 acts through several receptors. FPR2 (formyl peptide receptor 2, also called FPRL1) is a Gi-coupled class A GPCR expressed on neutrophils, monocytes and endothelial cells. LL-37 binding to FPR2 triggers pertussis toxin-sensitive calcium mobilisation, ERK1/2 phosphorylation and directed cell migration in transwell assays, and the response is blocked by the FPR2 antagonist WRW4. On keratinocytes and epithelial cells, LL-37 transactivates the epidermal growth factor receptor (EGFR): it stimulates metalloproteinase-dependent shedding of membrane-bound EGFR ligands, which then phosphorylate EGFR and drive ERK and STAT3 signalling and cell migration. On monocytes it opens the P2X7 ion channel, leading to caspase-1 activation and processing of pro-IL-1beta. On mast cells it activates MRGPRX2, a Gq-coupled receptor that triggers degranulation. LL-37 also binds DNA and RNA through its cationic face; the resulting complexes are taken up by plasmacytoid dendritic cells and activate endosomal TLR9 and TLR7, which is studied as a model of nucleic acid sensing.
Research applications
LL-37 sits in NuVion’s Tissue Repair Signalling category. Common in vitro uses include:
- Broth microdilution to determine minimum inhibitory and minimum bactericidal concentrations against reference strains of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, with low-salt and high-salt media run in parallel because divalent cations and serum lower its apparent activity
- Membrane permeabilisation assays: SYTOX Green or propidium iodide uptake in bacteria, calcein leakage from phosphatidylglycerol liposomes, and circular dichroism in SDS micelles or trifluoroethanol to follow helix formation
- Biofilm assays using crystal violet staining, including inhibition of attachment and dispersal of established biofilms at sub-MIC concentrations
- Lipopolysaccharide binding and neutralisation measured by the Limulus amoebocyte lysate assay and by TNF-alpha or IL-6 release from THP-1 cells
- FPR2 pharmacology in HEK293-FPR2 cells or differentiated HL-60 cells: calcium flux, transwell chemotaxis and receptor antagonist controls
- Keratinocyte and endothelial cell culture: scratch migration assays, phospho-EGFR and phospho-ERK western blots, and endothelial tube formation on basement membrane matrix, with LDH release or haemolysis assays to define the cytotoxic concentration range
Handling in the laboratory
Reconstitute the lyophilised powder with bacteriostatic water, adding the water gently and letting the solid dissolve without vigorous shaking. The reconstitution calculator gives the volume needed for a chosen stock concentration. Because LL-37 is strongly cationic and amphipathic it adsorbs to glass and to standard polystyrene, so low-protein-binding polypropylene tubes and plates are the usual choice, and dilution into high-salt buffers is done immediately before use to limit aggregation. Keep the sealed vial dry, away from light and refrigerated as described in the product documentation, and keep reconstituted solution refrigerated and use it within the period the documentation specifies. Each lot is characterised by RP-HPLC for purity and by mass spectrometry for identity, so the deconvoluted mass can be compared with the expected 4493 g/mol.
Testing and supply from NuVion
NuVion’s LL-37 comes from GMP-audited manufacture and is supplied lyophilised in sealed vials. Most batches are independently tested by Janoshik Analytical for purity by RP-HPLC and identity by mass spectrometry, and the Certificate of Analysis for a tested batch is published on the product page and in the COA library. Orders are dispatched from within Australia.
Related compounds
KPV, the C-terminal tripeptide of α-MSH, is studied in the same NF-kB and epithelial cell models, and Thymosin Alpha-1 is a 28-residue acetylated peptide used in Toll-like receptor and dendritic cell signalling work.
Frequently asked questions
What is LL-37 used for in research?
It is the standard human cathelicidin reference peptide. Microbiology groups use it for MIC, membrane permeabilisation and biofilm assays, biophysics groups use it as a model amphipathic helix for lipid bilayer studies, and cell biology groups use it as an FPR2 agonist and EGFR transactivator in migration and signalling assays.
Why does LL-37 activity change between media?
Its action on membranes depends on electrostatic binding, so sodium chloride, magnesium and calcium compete with the peptide for anionic headgroups and raise the measured MIC. Serum proteins such as apolipoprotein A-I bind the hydrophobic face and reduce free peptide. Assays are therefore reported with the medium stated, and low-salt and standard media are often run together.
Is LL-37 a therapeutic good in Australia?
No. LL-37 from NuVion is a laboratory chemical for in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the TGA. It is not for human or veterinary use.
How should LL-37 be stored?
Keep the lyophilised powder sealed, dry, away from light and refrigerated according to the product documentation. Once reconstituted with bacteriostatic water, keep the solution refrigerated in a low-binding tube and use it within the period stated in the documentation.
Research use only. This product is a laboratory chemical supplied for in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the Therapeutic Goods Administration for quality, safety or efficacy. It is not for human or veterinary use, and nothing on this page is a representation about therapeutic use.
